Abstract
Retinal pigment epithelial (RPE) cells that underlie the neurosensory retina are essential for the maintenance of photoreceptor cells and hence vision. Interactions between the RPE and their basement membrane, i.e. the inner layer of Bruch’s membrane, are essential for RPE cell health and function, but the signals induced by Bruch’s membrane engagement, and their contributions to RPE cell fate determination remain poorly defined. Here, we studied the functional role of the soluble complement regulator and component of Bruch’s membrane, Factor H-like protein 1 (FHL-1). Human primary RPE cells adhered to FHL-1 in a manner that was eliminated by either mutagenesis of the integrin-binding RGD motif in FHL-1 or by using competing antibodies directed against the α5 and β1 integrin subunits. The results obtained from primary RPE cells were replicated using the hTERT-RPE cell line. RNAseq expression analysis of hTERT-RPE cells bound to FHL-1 showed an increased expression of the heat-shock protein genes HSPA6, CRYAB, HSPA1A and HSPA1B when compared to cells bound to fibronectin (FN) or laminin (LA). Pathway analysis implicated changes in EIF2 signalling, the unfolded protein response, and mineralocorticoid receptor signalling as putative pathways. Subsequent cell survival assays using H2O2 to induce oxidative stress-induced cell death showed hTERT-RPE cells had significantly greater protection when bound to FHL-1 or LA compared to plastic or FN. These data show a non-canonical role of FHL-1 in protecting RPE cells against oxidative stress and identifies a novel interaction that has implications for ocular diseases such as age-related macular degeneration.
Competing Interest Statement
Paul Bishop, and Simon Clark are inventors of patent applications that describe the use of complement inhibitors for therapeutic purposes, and are co-founders and Directors of Complement Therapeutics, which is developing complement inhibitors for therapeutic purposes. All other authors declare that they have no conflicts of interest.
Abbreviations
- AMD
- Age-related macular degeneration
- CCP
- Complement control protein
- DMEM
- Dulbecco’s modified eagle’s medium
- ECM
- Extracellular matrix
- FCS
- Foetal calf serum
- FDR
- False discovery rate
- FH
- Factor H
- FHL-1
- Factor H-like protein 1
- FHR-4
- Factor H-related protein 4
- FI
- Factor I
- FN
- Fibronectin
- GAGs
- Glycosaminoglycans
- LA
- Laminin
- LAL
- Limulus amebocyte lysate
- MR
- Mineralocorticoid receptor
- NSR
- Neurosensory retina
- NGS
- Normal goat serum
- PFA
- Paraformaldehyde
- RPE
- retinal pigment epithelium